Structural Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.
Structural Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Howard Hughes Medical Institute, 1275 York Avenue, New York, NY 10065, USA.
Cell. 2018 Jun 14;173(7):1663-1677.e21. doi: 10.1016/j.cell.2018.05.041.
The ribonucleolytic RNA exosome interacts with RNA helicases to degrade RNA. To understand how the 3' to 5' Mtr4 helicase engages RNA and the nuclear exosome, we reconstituted 14-subunit Mtr4-containing RNA exosomes from Saccharomyces cerevisiae, Schizosaccharomyces pombe, and human and show that they unwind structured substrates to promote degradation. We loaded a human exosome with an optimized DNA-RNA chimera that stalls MTR4 during unwinding and determined its structure to an overall resolution of 3.45 Å by cryoelectron microscopy (cryo-EM). The structure reveals an RNA-engaged helicase atop the non-catalytic core, with RNA captured within the central channel and DIS3 exoribonuclease active site. MPP6 tethers MTR4 to the exosome through contacts to the RecA domains of MTR4. EXOSC10 remains bound to the core, but its catalytic module and cofactor C1D are displaced by RNA-engaged MTR4. Competition for the exosome core may ensure that RNA is committed to degradation by DIS3 when engaged by MTR4.
核糖核酸内切酶 RNA 外切体与 RNA 解旋酶相互作用以降解 RNA。为了了解 3' 到 5' Mtr4 解旋酶如何与 RNA 和核外切体结合，我们从酿酒酵母、裂殖酵母和人类中重新组装了含有 14 个亚基的 Mtr4 RNA 外切体，并表明它们可以解旋结构底物以促进降解。我们用人外切体加载了一种优化的 DNA-RNA 嵌合体，该嵌合体在解旋过程中使 MTR4 失活，并通过冷冻电镜（cryo-EM）确定其结构的整体分辨率为 3.45Å。该结构揭示了一个在非催化核心顶部结合 RNA 的解旋酶，RNA 被捕获在中央通道内，并且 DIS3 外切核酸酶活性位点。MPP6 通过与 MTR4 的 RecA 结构域接触将 MTR4 连接到外切体上。EXOSC10 仍然结合在核心上，但与其催化模块和辅助因子 C1D 被与 RNA 结合的 MTR4 取代。与外切体核心的竞争可能确保当 RNA 与 MTR4 结合时，它被 DIS3 降解。
